The present invention relates to antigen-toxin chimeric proteins useful in the targeted immunotherapy of autoimmune diseases, and particularly of multiple sclerosis.
The development of selective immunosuppressive agents is one of the major goals in the treatment of autoimmune diseases. In the ignorance of the identity of the specific antigen involved, treatment has until now been oriented toward nonspecific killing of rapidly dividing cells by means of cytotoxic agents, as well as inhibiting the action of mediators of inflammation with anti inflammatory agents.
More recently, specific and selective agents for the therapy of disorders of the immune response have been developed, based on our increased understanding of the immune response, advances in genetic engineering and improved models of autoimmune diseases.
The well studied animal model Experimental Autoimmune Encephalomyelitis (EAE) is known to be induced by Myelin Basic Protein (MBP) or its immunogenic determinants, in many mammalian species when injected under appropriate conditions. MBP is a major component of central nervous system (CNS) myelin proteins. It has been proposed that MBP stimulates populations of T cells causing them to migrate into the central nervous system and initiate a response that results in perivascular cuffing lesions and demyelination characteristic of multiple sclerosis. This model can also be induced by injecting (MIBP-specific -T helper cells into murine animals.
Many approaches have been taken to inhibit the EAE model. Monoclonal antibodies specific to the MBP-1a complex were reported to inhibit EAE in H-2S mice (1-2)
Other approaches to the treatment of multiple sclerosis (MS) include the administration of synthetic T-cell receptor peptides (3), altered peptides of MBP (4), copolymer-1xe2x80x94a synthetic random copolymer of amino acids (U.S. Pat. No. 3,849,550) and various interferons (5).
A different approach to the treatment of autoimmune diseases relates to the use of chimeric cytotoxic molecules that are produced by gene fusion techniques. These molecules utilize portions of toxins such as Pseudomonas exotoxin (PE) while eliminating its nonspecific cell binding properties. Specificity is added to the truncated or modified toxin molecules by fusion of the toxin moiety with a recognition element which directs the chimeric protein to selected target cells expressing a specific receptor.
Effective chimeric cytotoxins have been constructed by fusion of cDNA""s encoding IL2 IL4, IL6, TGFxcex1, anti Tac and CD4 with PE (6-11).
One of the chimeric proteins, IL2-PE40 has been designed to target and selectively eliminate activated T cells expressing IL2 receptors. IL2-PE40 was found to be an effective and selective immunosuppressive agent for IL2 receptor targeted therapy in many models of disorders of the immune response, where activated T cells play a crucial role (12-19).
Using a highly purified preparation (20), IL2-PE40 has been shown to (a) delay and mitigate adjuvant induced arthritis in rats (21), (b) significantly prolong the survival of vascularized heart allografts in mice (22), (c) reduce the incidence and severity of experimental autoimmune uveoretinitis (EAU) in rats, (23), (d) suppress the growth of a T cell lymphoma in mice (24), (e) significantly reduce the clinical rejection score and cumulative rejection rate in orthotopic corneal grafts in rats (25) and (t) prevent the characteristic features of EAE in rats and mice (26-27).
Although cytokine-toxin chimeric proteins such as IL2-PE40 have been shown to be an effective and selective immunosuppressive agent, its effects are not limited to specific antigen activated cells but rather involve all IL2 receptor positive cells (12).
In contrast to many strategies undertaken or proposed today to treat EAE or MS patients, there remains a need for a specific approach that targets only the pathogenic cells and will leave other immune responses fully operative.
Oligonucleotides coding for an MBP encephalogenic moiety fused to a cDNA encoding a truncated or mutated full length PE gene were disclosed by Steinberger I., et al (Third International Conference on Neuro-immunology, Jerusalem, Israel (1991), Third International Symposium on Immunotoxins Jerusalem (1992)). The DNA was expressed in E. Coli and the resulting chimeric proteins efficiently killed xcex1MBP T cells while having no effect on non-target cells.
It is an object of the present invention to provide a chimeric protein useful in the treatment of an autoimmune disease.
It is a further object of the present invention to provide a method of treatment of autoimmune diseases such as multiple sclerosis.
The present invention relates to the construction, characterization and purification of chimeric molecules comprising a myelin basic protein moiety linked to a toxin moiety that are efficient and selective novel agents to target and specifically kill xcex1MBP specific T cells, as well as peripheral blood cells from MS patients. According to one aspect of the present invention, there is provided a chimeric protein comprising a Pseudomonas aeruginosa exotoxin (PE) moiety linked to a myelin basic protein (MBP) moiety selected from the group comprising: (a) MBP; (b) amino acids 69-88 of guinea-pig myelin basic protein or an antigenic portion thereof; (c) amino acids 84-102 of human myelin basic protein or an antigenic portion thereof; (d) amino acids 143-168 of human myelin basic protein or an antigenic portion thereof; and (e) an amino acid sequence in which one or more amino acids have been deleted, added, substituted or mutated in the amino acid sequences of (a), (b), (c) or (d), the modified sequence retaining at least 75% homology with the unmodified amino acid sequences.
Analysis of the Pseudomonas exotoxin has revealed that it is comprised of several functionally distinct domains, referred to as domains I (a and b), II and III. Preferably, the Pseudomonas toxin moiety comprises the full length exotoxin or is a mutated or truncated derivative of said exotoxin. The exotoxin may be derived from domains II and III of the exotoxin. Alternatively, the Pseudomonas toxin moiety may be derived from a Pseudomonas exotoxin mutated, for example, at one or more of positions 57, 246, 247, and 249. Such a mutated PE is referred to as PE664Glu.
In an alternative embodiment the invention relates to a chimeric protein comprising a full length myelin basic protein linked to an optionally mutated or truncated Pseudomonas exotoxin moiety.
In either embodiment of the invention the myelin basic protein moiety and toxin moiety may be linked directly or indirectly. If the two moieties are indirectly linked then they are preferably joined by means of a linker sequence. As used herein the term xe2x80x9clinker sequencexe2x80x9d relates to any amino acid sequence comprising from 1 to 5 amino acids that may be repeated from 1 to 3 times, i.e. the linker may comprise up to 15 amino acid residues consisting of a pentapeptide repeated three times. Preferably the linker sequence is a pentapeptide comprising gly-gly-gly-gly-ser (SEQ ID NO:17).
A further aspect of the present invention relates to plasmids containing a DNA sequence encoding a molecule of the present invention and expression vectors capable of expressing said molecules. Suitable plasmids comprise a promoter operatively linked to a DNA sequence encoding a molecule of the present invention. Any prokaryotic promoter may be used such as PL, Tac and the like, preferably a bacteriophage T7 promoter is used. The term xe2x80x9coperatively linkedxe2x80x9d is used herein to mean that the promoter sequence and the sequence to be expressed under the control of said promoter are spatially positioned in respect of one another such as to permit the induction of expression by said promoter. Thus, the promoter and sequence to be expressed may be separated by means of a spacer sequence. Any suitable expression vector may be utilized, preferably, the vector used is E.coli. 
Also included in the scope of the present invention are salts of the described chimeric proteins. The term xe2x80x9csaltsxe2x80x9d includes both salts of carboxy groups as well as acid addition salts of amino groups of the protein molecule. Salts of the carboxy group may be formed by methods known in the art and include both inorganic salts such as sodium, calcium, ammonium, ferric or zinc salts and the like as well as salts with organic bases such as triethanolamine, arginine, lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as for example, hydrochloric acid, sulfuric acid or salts with organic acids such as acetic acid or oxalic acid.
The invention further relates to pharmaceutical compositions comprising at least one chimeric protein as defined above together with a pharmaceutically acceptable inert carrier. Preferably, the composition further includes a lysosomotrophic agent such as ammonium chloride, monensin and the like.
The proteins of the present invention may be administered by methods known in the art for the administration of proteins for example, oral, intravenous, intraarticular, sub-cutaneous, intramuscular, intra-peritoneal, intra-nasal, intrathecal intra-dermal, trans-dermal, by inhalation or any other route including the enteral route.
The chimeric protein of the present invention may be prepared by methods familiar to those skilled in the art for example, by chemical synthetic methods or by biotechnological (genetic) methods. If the latter is used a construct of the DNA encoding the chimeric protein is inserted into plasmid. A suitable vector is the selected and transformed by such a plasmid. The vector can then be stimulated to produce the desired chimeric protein.